Identifying key molecules that mediate brain reward plasticity remains an important goal of current drug abuse research. We recently identified a key role for MEF2 transcription factors as regulators of cocaine-induced synaptic and behavioral plasticity associated with repeated cocaine exposure. We find that cocaine-dependent inhibition of MEF2 in the NAc is required for increased MSN spine density, and that enhanced synaptic connectivity in the NAc after chronic cocaine exposure represents a compensatory mechanism that limits maladaptive behavioral responses associated with addiction, rather than supporting them. In this grant, we will elucidate cocaine- and cAMP-induced signaling events that control MEF2 activity in the striatum, including an exciting new regulatory mechanism for class IIa histone deacetylases that could have important implications for epigenetic regulation of drug addiction. To this end, we propose the following: Specific Aim 1: Our preliminary findings suggest that chronic cocaine exposure inhibits MEF2 activity by a cAMP-dependent process involving inhibitory phosphorylation of MEF2 (P-S408/444). In this aim, we will test the hypothesis that cocaine and cAMP signaling regulates MEF2 activity through control of P-S408/444 levels. In doing so, we will also characterize the spatial and temporal regulation of P-MEF2 in vivo after cocaine exposure. To these ends, we will use established experimental approaches, such as immunohistochemistry, RNAi-based protein replacement and transcriptional reporter assays, to test in vivo and in cultured primary striatal neurons the importance of P-S408/444 MEF2 for cocaine and cAMP-dependent regulation of MEF2. Specific Aim 2: Our preliminary findings revealed that overexpression of the regulator of calmodulin signaling (RCS) negatively regulates MEF2 activity in cultured striatal neurons in a Ser55 dependent manner (PKA site). In this aim, we will test the hypothesis that P-S55 RCS is required for cAMP-dependent inhibition of basal and calcium-activated MEF2-dependent transcription, and test the hypothesis that RCS functions in vivo to limit sensitized behavioral responses to cocaine. To this end, we will analyze existing RCS knockout mice using established experimental approaches to test in vivo, and in cultured striatal neurons, the importance of RCS as a MEF2 negative regulator and as a cocaine-induced regulator of behavioral plasticity in vivo. Specific Aim 3: Our preliminary studies indicate that activation of cAMP/PKA signaling promotes the nuclear import of HDAC5 in striatal neurons via dephosphorylation of HDAC5 at a novel Cdk5 site. In this aim, we will test the hypothesis that chronic cocaine exposure, via PKA-dependent dephosphorylation of HDAC5, promotes enhanced HDAC5 nuclear localization, which serves to reduce transcriptional responses and limit sensitized behavioral responses to repeated cocaine exposure. We will test this idea using novel P-HDAC5 antibodies with NAc tissues of cocaine exposed mice, mechanistic analysis of HDAC5 nucleocytoplasmic shuttling, and behavioral analysis of HDAC5 phospho-site mutant-expressing mice.